Project Description TNF?/TNFR has received the greatest attention because of its position at the apex of the pro-inflammatory cy- tokine cascade, and its dominance in the pathogenesis of inflammation. TNFR1 primarily mediates inflammato- ry activity of TNF?, whereas TNFR2 plays a protective and anti-inflammatory role in various diseases. Our ge- netic screen for the binding partners of progranulin (PGRN) growth factor led to the isolation of TNFR2 as the PGRN-binding receptor (Tang, et al, Science, 2011). Remarkably, PGRN exhibits an approximately 600-fold higher binding affinity to TNFR2 than does TNF?. During the initial funding period, we have successfully identi- fied in a proteomics screen 14-3-3?, an important intracellular signaling molecule, as a novel component of TNFR2 complexes in response to PGRN stimulation. PGRN deficiency promoted inflammatory classically- activated macrophage (CAM) but inhibited anti-inflammatory alternatively-activated macrophage (AAM) polari- zation, whereas recombinant PGRN inhibited AAM-CAM but stimulated CAM-AAM switching. In addition, knockout of 14-3-3? abolished PGRN?s regulation of macrophage. More excitingly, our direct protein-protein interaction screen of nine central members of the complement system identified C5a as a novel binding partner of PGRN with high binding affinity, and PGRN inhibited the binding of C5a to C5aR1. Thus, the scientific prem- ise of this competitive continued application is based on 1) the identification of 14-3-3? as a novel component of PGRN/TNFR2 pathway, and 2) the isolation of C5a as a novel PGRN-binding factor. The central hypothesis is that PGRN regulates macrophage polarization and inflammatory arthritis through a) recruitment of 14-3-3? to TNFR2 and activation of the PGRN/TNFR2 anti-inflammatory pathway; and b) interplay with C5a and inhibition of C5a/C5aR1 inflammatory pathway. The Specific Aims are: (1) To elucidate 14-3-3??s role in PGRN/TNFR2- mediated macrophage polarization in the course of inflammatory arthritis. We will determine the dependence on 14-3-3? of PGRN's regulation of macrophage polarization (SA#1.1); the signaling, target genes and co- factor(s) of 14-3-3? that mediate PGRN action in macrophage polarization (SA#1.2); and the importance of PGRN regulation of macrophage phenotypic switch in the context of inflammatory arthritis and its dependence on 14-3-3? (SA#1.3). (2) To define the importance of PGRN/C5a interplay in the pathogenesis of inflammatory arthritis. We will characterize and dissect the PGRN/C5a interaction and to identify PGRN-derived minimal fragment/peptide that retains C5a binding activity (SA#2.1); elucidate the interplay between PGRN and C5a in regulating macrophage polarization, as well as the signaling pathways involved (SA#2.2); and determine the interplay between PGRN and C5a/C5aR1 signaling and the contribution of inhibition of C5a/C5aR1 signaling by PGRN to PGRN's anti-inflammatory action in inflammatory arthritis (SA#2.3). The proposed research will not only advance our understanding of the pathogenesis of inflammatory arthritis, but may also lead to the de- velopment of new interventions for various TNFR- and C5a/C5aR1-related conditions and diseases.